Histidine modifying agents abolish pyruvate dehydrogenase kinase activity

Pyruvate dehydrogenase kinase (PDK) specifically phosphorylates the E1 alph a subunit of the pyruvate dehydrogenase complex (PDC). Sequence analysis of cloned PDKs led to the proposal that they are mechanistically related to p rokaryotic 2-component His-kinases. The reaction mechanism of protein His-k inases involves autophosphorylation of a specific His residue followed by p hosphotransfer to an Asp residue. Treatment of recombinant Arabidopsis thal iana PDR with the His-directed reagents diethyl pyrocarbonate (DEPC) and di chloro-(2,2':6',2''-terpyridine)platinum(II) dihydrate led to a marked inhi bition of autophosphorylation. In addition, DEPC treatment abolished the ab ility of PDK to trans-phosphorylate and inactivate PDC. These results valid ate the prediction that PDKs require His residues for activity.